Motifs of Two Small Residues can Assist but are not Sufficient to Mediate Transmembrane Helix Interactions (original) (raw)
Related papers
Journal of Biological Chemistry, 2004
Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXG-VXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and ϳ18 Å in structural space), work in concert to control TM-TM affinity synergistically.
The GxxxG motif: A framework for transmembrane helix-helix association 1 1 Edited by G. von Heijne
J Mol Biol, 2000
In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-af®nity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A. Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane. Strong selection was applied to a large range of sequences ($10 7 possibilities) and resulted in the identi®cation of sequence patterns that mediate high-af®nity helix-helix association. The most frequent motif isolated, GxxxG, occurs in over 80 % of the isolates. Additional correlations suggest that¯anking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identi®ed sequence patterns represent packing motifs. The convergent identi®cation of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins.
The GxxxG motif: A framework for transmembrane helix-helix association
Journal of Molecular Biology, 2000
In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-af®nity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A. Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane. Strong selection was applied to a large range of sequences ($10 7 possibilities) and resulted in the identi®cation of sequence patterns that mediate high-af®nity helix-helix association. The most frequent motif isolated, GxxxG, occurs in over 80 % of the isolates. Additional correlations suggest that¯anking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identi®ed sequence patterns represent packing motifs. The convergent identi®cation of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins.
Specificity and promiscuity in membrane helix interactions
FEBS Letters, 1994
Transmembrane a-helices can associate with one another in lipid bilayers. This association is important in the folding and oligomerization of many integral membrane proteins, and may also play a role in their function. The interactions between helices may be highly specitic or relatively non-specific, and their roles may differ accordingly. These two cases are discussed.
Association of transmembrane helices: what determines assembling of a dimer?
Journal of Computer-Aided Molecular Design, 2006
Self-association of two hydrophobic a-helices is studied via unrestrained Monte Carlo (MC) simulations in a hydrophobic slab described by an effective potential. The system under study represents two transmembrane (TM) segments of human glycophorin A (GpA), which form homo-dimers in membranes. The influence of TM electrostatic potential, thickness and hydrophobicity degree of lipid bilayer is investigated. It is shown that the membrane environment stabilizes a-helical conformation of GpA monomers, induces their TM insertion and facilitates inter-helical contacts. Head-to-head orientation of the helices is promoted by the voltage difference across the membrane. Subsequent ''fine-tuned'' assembling of the dimer is mediated by van der Waals interactions. Only the models of dimer, calculated in a hydrophobic slab with applied voltage agree with experimental data, while simulations in vacuo or without TM voltage fail to give reasonable results. The moderate structural heterogeneity of GpA dimers (existence of several groups of states with close energies) is proposed to reflect their equilibrium dynamics in membrane-mimics. The calculations performed for GpA mutants G83A and G86L permit rationalization of mutagenesis data for them. The results of Monte Carlo simulations critically depend on the parameters of the membrane model: adequate description of helix association is achieved in the water-cyclohexane-water system with the membrane thickness 30-34 Å , while in membranes with different hydrophobicities and thickness unrealistic conformations of the dimer are found. The computational approach permits efficient prediction of TM helical oligomers based solely on the sequences of interacting peptides.
Specificity in Transmembrane Helix-Helix Interactions Mediated by Aromatic Residues
Journal of Biological Chemistry, 2007
The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alaninescanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several ''hot spots'' for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design.
Structural Determinants of Transmembrane Helical Proteins
Structure, 2009
We identify a structural feature of transmembrane helical proteins that restricts their conformational space and suggests a new way of understanding the construction and stability of their native states. We show that five kinds of well-known specific favorable interhelical interactions (hydrogen bonds, aromatic interactions, salt bridges, and two interactions from packing motifs) precisely determine the packing of the transmembrane helices in 15 diverse proteins. To show this, we iteratively reassemble the helix bundle of each protein using only these interactions, generic interaction geometries, and individual helix backbone conformations. On average, the representative set of rebuilt structures best satisfying the constraints imposed by the five types of interhelical interactions has an average Ca root-mean-square deviation from the native of 1.03 Å . Implications for protein folding, structure and motion prediction, modeling, and design are discussed.
A sequence and structural study of transmembrane helices
Journal of computer-aided molecular design, 2001
A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the beta-branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline. residue types normally associated with 'helix-breaking' propensity are found t...
J Mol Biol, 2000
To ®nd motifs that mediate helix-helix interactions in membrane proteins, we have analyzed frequently occurring combinations of residues in a database of transmembrane domains. Our analysis was performed with a novel formalism, which we call TMSTAT, for exactly calculating the expectancies of all pairs and triplets of residues in individual sequences, taking into account differential sequence composition and the substantial effect of ®nite length in short segments. We found that the number of sig-ni®cantly over and under-represented pairs and triplets was much greater than the random expectation. Isoleucine, glycine and valine were the most common residues in these extreme cases. The main theme observed is patterns of small residues (Gly, Ala and Ser) at i and i 4 found in association with large aliphatic residues (Ile, Val and Leu) at neighboring positions (i.e. i AE 1 and i AE 2). The most over-represented pair is formed by two glycine residues at i and i 4 (GxxxG, 31.6 % above expectation, p < 1 Â 10 À33 ) and it is strongly associated with the neighboring b-branched residues Ile and Val. In fact, the GxxxG pair has been described as part of the strong interaction motif in the glycophorin A transmembrane dimer, in which the pair is associated with two Val residues (GVxxGV). GxxxG is also the major motif identi®ed using TOX-CAT, an in vivo selection system for transmembrane oligomerization motifs. In conjunction with these experimental observations, our results highlight the importance of the GxxxG b-branched motif in transmembrane helix-helix interactions. In addition, the special role for the b-branched residues Ile and Val suggested here is consistent with the hypothesis that residues with constrained rotameric freedom in helical conformation might reduce the entropic cost of folding in transmembrane proteins. Additional material is available at
Glycophorin A Dimerization Is Driven by Specific Interactions between Transmembrane a-Helices
1992
Specific side-by-side interactions between transmembrane a-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane a-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane a-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the a-helix disrupt dimerization, suggesting a high degree of specificity in the helixhelix interactions. This approach allows the interface between interacting helices to be defined.